一贯煎联合骨髓间充质干细胞调控RhoA/ROCK1通路抑制大鼠肝纤维化的实验研究
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国家自然科学基金项目(81573879)


Yiguanjian Combined with Bone Marrow Mesenchymal Stem Cells Treats Liver Fibrosis in Rats via RhoA/ROCK1 Signaling Pathway
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    摘要:

    目的:探究一贯煎联合骨髓间充质干细胞在治疗肝纤维化过程中对RhoA/ROCK1通路的作用。方法:将72只SD大鼠随机分为正常对照组、模型对照组、秋水仙碱组、一贯煎高剂量组、一贯煎中剂量组、一贯煎低剂量组、干细胞组、一贯煎+干细胞组;四氯化碳腹腔注射6周制备肝纤维化动物模型,干预4周后取材。记录大鼠阴虚表征情况;检测血清谷丙转氨酶(GPT)、谷草转氨酶(GOT)含量;HE、Masson染色观察病理改变;实时PCR、蛋白质印迹法检测肝组织中RhoA、Rho相关激酶1(ROCK1)、α-SMA和COL-1 mRNA及蛋白表达;IHC检测α-SMA、COL-1阳性面积。结果:与正常对照组比较,模型对照组大鼠具有明显阴虚表征,肝脏出现明显炎症浸润和纤维增生,肝组织RhoA、ROCK1、α-SMA、胶原蛋白-1(COL-1)mRNA与蛋白表达显著升高(P<0.05),α-SMA、COL-1阳性面积显著增加(P<0.05);与模型对照组比较,一贯煎高剂量组、一贯煎中剂量组、一贯煎低剂量组、骨髓干细胞、一贯煎+干细胞组大鼠的阴虚表征、肝脏炎症和纤维化程度减轻,RhoA、ROCK1、α-SMA和COL-1 mRNA与蛋白表达显著降低(P<0.05),α-SMA、COL-1阳性面积显著减少(P<0.05),其中一贯煎+干细胞组较一贯煎中剂量组、干细胞组效果更优(P<0.05)。结论:一贯煎与骨髓干细胞可通过调控RhoA/ROCK1通路抑制肝星状细胞活化从而发挥抗纤维化的作用,二者协同作用效果优于各自单独作用效果。

    Abstract:

    To observe the effect of Yiguanjian(YGJ) combined with bone marrow mesenchymal stem cells(BMSCs) on Ras homolog gene family,member A(RhoA)/Rho-associated coiled-coil containing protein kinase 1(ROCK1) signaling pathway in the treatment of liver fibrosis.Methods:Seventy-two SD rats were randomly assigned into a normal group,a model group,a colchicine group,high-,medium,and low-dose YGJ(YGJ-H,YGJ-M,and YGJ-L,respectively) groups,a BMSCs group,and a YGJ+BMSCs group.The rat model of liver fibrosis was established by intraperitoneal injection of carbon tetrachloride(CCl4) for 6 weeks,and the rats were sacrificed after 4 weeks of treatment.Before the rats were sacrificed,we scored their behavior of yin deficiency.We then measured the content of glutamic-pyruvic transaminase(GPT) and glutamic-oxaloacetic transaminase(GOT) in the serum and stained the liver with hematoxylin-eosin(HE) and Masson to observe the pathological changes.RT-PCR and Western blotting were respectively employed to determine the mRNA and protein levels of RhoA,ROCK1,α-smooth muscle actin(α-SMA),and collagen type I(COL-1).Immunohistochemistry(IHC) was adopted to detect α-SMA and COL-1 positive areas in liver tissue sections.Results:Compared with the normal group,the modeling of liver fibrosis elevated the behavior scores of yin deficiency,caused liver inflammation and fibroplasia,up-regulated the mRNA and protein levels of RhoA,ROCK1,α-SMA,and COL-1(P<0.05),and increased the positive areas of α-SMA and COL-1(P<0.05).Compared with the model group,YGJ-H,YGJ-M,YGJ-L,BMSCs,and YGJ+BMSCs relieved the behaviors of yin deficiency,liver inflammation,and fibrosis(P<0.05),down-regulated the mRNA and protein levels of RhoA,ROCK1,α-SMA,and COL-1(P<0.05),and decreased the positive areas of α-SMA and COL-1(P<0.05).Among different treatment groups,YGJ+BMSCs group demonstrated the best performance(P<0.05).Conclusion:YGJ and BMSCs can inhibit the activation of hepatic stellate cells by inhibiting RhoA/ROCK1 signaling pathway to treat liver fibrosis,and their combination has better performance than YGJ or BMSCs alone.

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李汶航,张睿,崔欣怡,唐佐青,油红捷,杨铮,付修文,马赟,刘文兰.一贯煎联合骨髓间充质干细胞调控RhoA/ROCK1通路抑制大鼠肝纤维化的实验研究[J].世界中医药,2022,(18).

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  • 收稿日期:2021-08-02
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  • 在线发布日期: 2022-10-28
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